1. Field of the Invention
The present invention relates to a method for assaying an antiphospholipid antibody and a kit for use in the method. More particularly, the present invention relates to a method for assaying an antiphospholipid antibody and a kit therefor characterized by using, in place of .beta.2-glycoprotein I, a polypeptide containing the same amino acid sequence as a specific domain of .beta.2-glycoprotein I, or a polypeptide different in the amino acid sequence from the specific domain but functionally equivalent thereto.
2. Description of Related
Various assay methods including radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) for an anticardiolipin antibody, which is part of the antiphospholipid family, have been reported by Harris et al., Lancet, iii: 1211, 1983; Koike et al., Clin. Exp. Immunl., 56: 193, 1984; and the like.
However, those methods are not necessarily satisfactory, since they involve problems that an anticardiolipin antibody cannot be quantitatively assayed with a high accuracy, or that an anticardiolipin antibody from patients with infectious diseases cannot be assayed differentially from these patients with antiphospholipid syndrome.
Recently, Matsuura et al. have discovered that an anticardiolipin antibody from patients with antiphospholipid syndrome does not recognize an immobilized cardiolipin but a complex of cardiolipin and .beta.2-glycoprotein I (.beta.2-GPI, also called apolipoprotein H or anticardiolipin cofactor). Further, they have developed a new assay for the antiphospholipid antibody based on the new findings, which can overcome the prior art problems [Lancet, 336: 177, 1990; RINSHO MEN-EKI (Clinical Immunology), 22 (Suppl. 15): 170, 1990; WO 91/06006; and J. Immunol., 148:3855, 1992].
Further studies according to Matsuura et al. have revealed that an autoantibody (hitherto called an anticardiolipin antibody) from patients with antiphospholipid syndrome does not react with cardiolipin itself in the complex of cardiolipin and .beta.2-GPI but recognize an altered .beta.2-GPI, of which structure has been conformationally changed due to interaction with a hydrophobic surface on which an oxygen atom is present, such as a cardiolipin-immobilized plate [J. Exp. Med., 179, 457-462 (1994); and KANSEN, ENSHO, MEN-EKI (Infection, Inflammation and Immunity), 23, 9 (1993)]. When applying this new principle, it is unnecessary to use a phospholipid such as cardiolipin as used in the prior art assays. Thus, Matsuura et al. have developed a new method for assaying an autoantibody from patients with antiphospholipid syndrome using a solid phase reagent wherein .beta.2-GPI alone is immobilized on a carrier with the surface on which a polar group has been introduced (WO 93/16387).
On the other hand, .beta.2-GPI is a known glycoprotein, and its amino acid sequence and nucleotide sequence have been already clarified [Int. Immunol., 3, 1217-1221 (1991); Biochem. J., 277, 387 (1991); and Gene, 108, 293 (1991)]. However, it has been considered that it is difficult to express and produce an active .beta.2-GPI according to a conventional expression system in E. coli or yeast, since processing and modification such as a formation of disulfide bond are not correctly proceeded for the following reasons:
(1) a native .beta.2-GPI has eleven cysteine-cysteine (S-S) bonds in the molecule, and has a characteristic primary structure called "sushi domain" composed of five domains as shown on FIG. 1; and PA1 (2) a nucleotide sequence encodes .beta.2-GPI as a precursor protein wherein a mature protein additionally has a peptide composed of 19 amino acids at the N-terminus, and such an additional peptide should be cleaved when secreted. PA1 (1) a phospholipid binding site is present in the fifth domain (domain V) of .beta.2-GPI as shown in FIG. 1; PA1 (2) an epitope (an antibody recognition site), which an autoantibody from patients with antiphospholipid syndrome recognizes, is present in a region centering around the fourth domain (domain IV); PA1 (3) this epitope is usually cryptic, with domain V binding to a phospholipid or the like, resulting in that the .beta.2-GPI molecule undergoes a conformational change, whereby the epitope is exposed to be recognized by the autoantibody.
However, Igarashi et al. have recently reported that a baculovirus expression system can overcome the difficulty as discussed above, and that a recombinant .beta.2-GPI produced by the baculovirus expression system can be utilized for an assay of an autoantibody from patients with antiphospholipid syndrome [Clin. Exp. Immunol., 93, 19 (1993); and Japanese Patent Application Serial No. 4-152619].
As stated hereinabove, it has been clarified that .beta.2-GPI is undoubtedly a protein necessarily required for an assay of an autoantibody from patients with antiphospholipid syndrome. Nevertheless, clinical significance of the autoantibody from patients with antiphospholipid syndrome still remains unclear. Accordingly, it is considered as being an important problem to be solved in the future to clarify the reactivity of the autoantibody from patients with antiphospholipid syndrome with .beta.2-GPI, which may result in a clarifying mechanism regarding how antiphospholipid syndrome is a caused, and may result in establishing a more convenient assay system.